PROJECT SUMMARY Recombinant adeno-associated viral (rAAV) vectors have emerged as one of the preferred delivery agents for clinical gene therapy. However, the gene therapy for cystic fibrosis lung disease has been hindered by the inefficiency of rAAV transduction from airway lumen. Using the in vitro model of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI), we found the transduction polarity of rAAV2 and rAAV5 that prefers basolateral infection. In spite of the apical transduction is low, over a thousand-fold augmentation can be achieved when the cellular proteasome activity is transiently inhibited. rAAV2.5T is a novel AAV vector developed from the directed evolution of a capsid library of shuffled AAV2 and AAV5 cap genes. rAAV2.5T demonstrates altered transduction polarity with an efficiency of apical transduction over 10-fold higher than those of rAAV2 and rAAV5 in polarized HAE, but its responses to proteasome inhibition still retain, suggesting that vector intracellular trafficking remains the major post-entry barrier in transduction. Currently, the mechanisms underlying the differences in the polarity of AAV transduction and the inefficient post-entry trafficking toward productive transduction in polarized HAE remain unclear. Recently, a type I transmembrane protein KIAA0319L, denoted hereafter as AAV receptor (AAVR), was identified as a proteinaceous receptor for certain AAV serotypes, including AAV2 and AAV5, but not all. Importantly, AAV2.5T transduces HeLa cells in the manners of both AAVR-dependent and independent entries. In polarized HAE, AAVR expresses only on basolateral membrane. While AAVR is responsible for the basolateral infections of rAAV2 and rAAV2.5T, a non-AAVR proteinaceous receptor for apical entry has not yet identified. We hypothesize that transduction polarity of AAV in polarized HAE is determined by the polarized expression of an alternative (non-AAVR) receptor on apical membrane and the AAVR on basolateral membrane, which divert the internalized vectors to different intracellular trafficking pathways depending on apical or basolateral endocytosis. In this project, we aim to identify the proteinaceous receptor that mediates apical infection of AAV2.5T, and to reveal vector entry and intracellular trafficking of this AAVR-independent transduction pathway, in comparison with the AAVR- mediated transduction from the basolateral membrane. We will also investigate how transient inhibition of the proteasome activity reroutes the internalized vectors toward a pathway prone to productive transduction. The preclinical studies of rAAV2.5T for cystic fibrosis (CF) gene therapy in CF ferret models are ongoing, the outcomes of this proposal will benefit the development of an effective vector delivery approach to use this vector in the gene therapy of human airway diseases, including CF.